The object of this project is to use the bacteriophage phi 29 as a model system to investigate the mechanism of replication of linear DNA molecules. First, we will determine the origin of replication of phi 29 DNA by labeling replicating DNA molecules with a short pulse and by determining the distribution of radioactivity in the DNA fragments generated by restriction endonucleases. Second, we will attempt to isolate and characterize replicative intermediates of phi 29 DNA from infected Bacillus subtilis cells. Third, we will study the nature of phi 29 DNA-protein association in detail using a variety of enzymes, such as exonucleases and DNA polymerases. Fourth, we will isolate phi 29 DNA terminal protein from infected cells to define the role in DNA replication. Finally, we propose to develop an in vitro DNA synthesis system from phi 29 infected cells to investigate the molecular mechanism of linear DNA replication.